Normal histologic procedures that are used to prepare slides bearing thin slices of tissues or other biological materials for microscopic examination require the use of so-called clearing solvents in at least two different stages of the processing:
1. To replace (dissolve away) alcohol and/or other dehydrants that are present in gross pieces of tissues immediately following tissue fixation steps. This replacing (clearing) solvent must also have the property of being readily soluble in the paraffin wax that is used in subsequent steps to "embed" the tissue.
2. To dissolve away the embedding paraffin wax that is present in the thin slices (sections) of tissue that are prepared with a microtome knife from the gross pieces (chunks) of tissues described above. The "dissolving" process must be rapid and complete, and the solvent must be easily removed by alcohol or other dehydrants that are used in subsequent washing steps.
Historically, the main solvents used for the "clearing" steps have been toluene and xylene. However, both of these solvents suffer from major problems including fire safety (relatively low flash- and boiling points) and multiple toxicities (including headaches, nausea, and damage to liver, kidneys, blood, eyes and nervous system), as well as being extremely noxious to work with in confined areas with limited air circulation like hospitals and laboratories.
In recent years, there have been numerous attempts to replace these solvents with safer and less-noxious types of clearing solvents, but these attempts largely have been unsuccessful. As a result of these unsuccessful attempts, it was generally thought that the effectiveness of solvents (for dewaxing purposes) was directly linked to their dangerous and noxious properties. That is, it was thought that the better the clearing solvent, the more dangerous and more noxious it was. The following is a brief summary of alternate clearing agents that have been used conventionally:
1. Chlorinated hydrocarbons are effective clearing solvents, but they are considered to be very toxic and noxious chemicals. Essentially, the use of all of the effective solvents has been restricted to some extent by the government.
2. Terpenes (including d-limonene, .alpha.-pinenes and dipentene) are moderately effective clearing solvents, but they too are considered toxic and reportable under OSHA. More critically, they are extremely noxious and overpowering to laboratory workers. They also tend to dry slowly and leave an oily residue.
3. Higher molecular weight paraffinic (straight-chain) and isoparaffinic (branched chain) hydrocarbon solvents generally are safer and less noxious than toluene and xylene, but they are not very good as clearing solvents. They not only dissolve wax quite slowly, as compared to xylene for example, but they also dry slowly and often leave an oily residue. Lower molecular weight paraffinic and isoparaffinic solvents have serious problems with fire safety and smell.
4. Aromatic petroleum-derived solvents generally are much more effective clearing solvents than the paraffinic and isoparaffinic materials, but they are very much more toxic and noxious. As noted above, effectiveness was generally considered to be related directly to toxicity, noxious odor, and flammability.